bmebt_phd_notesfandomcom-20200214-history
Operation of a Bioreactor
Outlined here are procedures and best practices for operation the RALF bioreactor, and on the design of fermentation experiments generally. General Bioreactor Features "Basic fermentation technology is an extension of the simple shake flask technique for growing cultures. It grew out of the desire to control growth environments for live cultures in a more complete and quantitative way. Batch culture shake flasks are usually limited by imprecise control of temperature. Temperature uniformity in an incubated shaker or warm room is highly variable, sometimes straying 5 °C or more from the intended setpoint. Since the shake flask is normally agitated at a fixed speed, oxygen uptake and gas exchange is limited. Once the available ambient oxygen is depleted, most cultures fail to thrive. There is no pH control in shake flasks. In many cases, if the culture is not limited by feed stock, it becomes acidic to the point of detriment to the culture and respiration slows dramatically. Most shake flask cultures are also run as a 'batch', which means that they are fed only once at or near the beginning of the cultures inoculation. After this initial carbon source is consumed, the culture stops growing. In some cases its metabolism may shift and begin to consume other metabolites in the culture broth, sometimes changing the characteristics of the resultant biomass or protein. Shake flasks are also usually subject to media evaporative loss in warmer culture environments, typically 10% of volume per 24 hr at 37 °C. This loss changes the density of the culture and prohibits longer term operation of the system. Finally, the user may encounter foaming from the media after agitation. The occurrence of foam in the headspace above the culture will limit gas exchange and further stifle growth. The basic fermentation system is designed to address all of these limitations. Careful temperature control is achieved in fermentation vessels by the use of impeller agitation and a heating jacket. A sensor inserted into the vessel and feedback control of heating and cooling of this jacket usually results in temperature control ±0.1 °C around the setpoint. Benchtop fermentors generally provide control of pH via liquid reagent addition through a pump. The pH value is continuously monitored in an effort to keep the environment optimal for cell growth. Proper aeration is maintained by the aforementioned mixing impeller or by the infusion of air or oxygen supplemented gas directly into the culture. With shear-sensitive cultures, oxygen supplemented gas is the primary mechanism for maintenance of oxygen level in the culture. Measurement of oxygen in solution is usually achieved by a polarographic probe which is not normally available for use in shake flasks. It is also possible to continuously or periodically add feed to the vessel to maintain growth in a linear or exponential fashion. The exit gas condenser provides a cold surface for vapor in the exhaust gas flow to condense, thus preserving culture volume and density. Periodic addition of antifoam surfactant is actuated by a conductivity probe in the culture, reducing foam on the surface and allowing gas exchange. The vessel, with all probes, fittings, impellers, harvest pipes, and tubes, is assembled and sterilized in a standard autoclave. After final probe calibrations and stabilization to operating environment, the culture is added to the vessel. The system can then be used to characterize the culture in a way that is more quantitative and precise than with a shake flask method. Tight control of temperature, pH, oxygen content, feed consumption, liquid evaporation, and foam levels all contribute to a much higher biomass and better protein yield." Obom KM, Magno A, Cummings PJ. Operation of a Benchtop Bioreactor. Journal of Visualized Experiments : JoVE. 2013;(79):50582. doi:10.3791/50582. Experimental Design with R. eutropha From El-Sayed, et al.